Many diagnostic assays utilize detectable labels to indicate binding events that that are indicative of the presence or absence of a target analyte in a sample. Typical target analytes include proteins, carbohydrates, or nucleic acids. Generally, such diagnostic assays utilize a target-specific capture molecule that is immobilized on a solid substrate. A sample is placed on the solid substrate and the target analyte, if present, binds to a target-specific capture molecule. The surface-bound target analyte may then be directly modified by binding, directly or indirectly, with a detectable label. Alternatively, a second reagent, modified with a detectable label, may bind to the surface immobilized target. The label can be detected directly in the case of radio-labeled or fluorescent labels using devices such as a phosphor-imager or a fluorescence reader, respectively. Alternatively, the label may be indirectly detected, for example, by binding the label with an anti-label/enzyme conjugate that is subsequently contacted with an enzyme substrate to produce a signal that can be detected.
Due to the low frequency of target analytes in some samples, various methods have been developed to enhance the signal of diagnostic assays using indirect methods. For example, U.S. Pat. No. 5,196,306, discloses a method in which a target-specific, surface-immobilized label is reacted with an amplification polymer to multiply the number of binding sites for a detectable label complex, followed by conjugation with an anti-label antibody conjugate, such as horse radish peroxidase (“HRP”) that is then exposed to a tyramide/label conjugate. The tyramide is activated by HRP and then reacts with electron rich groups nearby to physically attach a label molecule.
Several nucleic acid specific techniques have also been developed. For example, U.S. Pat. No. 5,124,246 discloses amplification of a signal by creating branched layers of DNA hybridization in a target nucleic acid sequence specific manner. The layers culminate in a branched structure that can hybridize to hundreds of labels. Other approaches, disclosed in U.S. Pat. No. 6,103,474 and U.S. Pat. No. 6,110,682, amplify a signal by targeting homopolymeric regions of a target nucleic acid analyte with multiple-labeled hairpin reporter probes. A method has also been developed that amplifies biotin-dependent signaling events (Zhong et al., PNAS (2003) 100:11559-11564). In this approach, biotinylated probes are immobilized on a surface in a target-dependent manner, and are then contacted with an avidin-biotinylated dextran copolymer, resulting in a 50-100 fold increase in assay sensitivity.
DNA dendrimers have also been used to amplify signals, as disclosed in U.S. Pat. No. 5,175,270, U.S. Pat. No. 5,487,973, and U.S. Pat. No. 6,046,038. DNA dendrimers are large cross-linked structures that can be modified to contain up to several hundred label groups. These labels groups include biotin, HRP, streptavidin (“SA”), and fluorescent molecules, as disclosed in U.S. Pat. No. 6,072,043; U.S. Pat. No. 6,110,687; and U.S. Pat. No. 6,762,292. DNA dendrimer can contain mixtures of molecules as well, such as SA and HRP. The mixture allows for binding of SA to surface-immobilized biotin, for example. This approach multiplies the number of HRP molecules at the surface of each biotin molecule bound and results in amplification of the signal.
Another technique employs the targeting of homopolymeric regions of target DNA with multiply-labeled hairpin reporter probes, as disclosed in U.S. Pat. Nos. 6,103,474; 6,110,682.
A method was recently disclosed that describes the amplification of biotin-dependent signaling events (Zhong et al., PNAS (2003) 100:11559-11564). Biotinylated probes that were immobilized onto a surface in a target-dependent manner were contacted with an avidin-biotinylated dextran copolymer. This was reported to increase assay sensitivity 50-100 fold increase in assay sensitivity for detection of biotin DNA probes covalently immobilized onto a chip surface. However, it has been observed that this method suffers from some inconsistency and high levels of non-specific interaction between the avidin-biotinylated dextran copolymer and the surface immobilized DNA probes resulting in an improvement in assay sensitivity of only 5-25 fold.
There continues to be a need for improvement in assays for detecting target nucleic acid analytes that may be present in samples.